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A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets
Introduction
Calcium flux assays are preferred methods in drug discovery for screening F protein coupled recepters (GPCRs) Screen Quest™ Fluo-8® No Wash Calcium Assay Kits provides homogeneous fluorescence-based assays for detecting intracellular calcium mobilization across a broad spectrum of biological targets. The characteristics of its long wavelength, high sensitivity and 200 times fluorescence enhancement (when it forms a complex with calcium) make Fluo-8® NW an ideal indicator for measurement of cellular calcium.
Fig. 1
Screen Quest™ Fluo-8 Calcium Assay
The signal intensity and well-to-well uniformity of Fluo-8® NW is compared with Fluo-3 AM, Fluo-4 NW yields the brightest signal, more the 2 fold brighter than Fluo-4 AM, and 4 times brighter than Fluo-3 AM. The Screen Quest™ Fluo-8® NW Calcium Assay Kits provide an optimized assay method for monitoring GPCRs and calcium channels. Unlike Fluo-3 AM and Fluo-4 AM, both of which require 37°C for optimal cell loading, the Screen Quest™ Fluo-8® NW requires a less temperature-dependent cell loading property, giving similar results either at room temperature or 37°C. This characteristic makes the Screen Quest™ Fluo-8® NW Calcium Assay Kits more robust for HTS applications.
Materials and Methods
- CHO-M1 or HEK cells were plated at 96-well (for NOVOstar) or 384-well black wall/clear bottom costar plate (for FLIPR) at 37°C incubator for overnight.
- Take growth medium off (if cells were plating in 0.51% FBS, skip this step), incubate the cells with Fluo-3 AM, Fluo-4 AM or Fluo-8® NW at room temp for 1-2 hr (or at 37°C for 30 min, then at room temp. for 30 min).
- For wash experiments: wash cells with HHBS buffer twice, then replace with HHBS buffer.
- For No wash experiments: run the experiments directly.
- Run calcium efflux experiments on NOVOstar or FLIPR
Fig. 2
Step-by-step process
Results
Fig. 3
U2OS cells were seeded overnight at 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µL of 4 µM Fluo-3 AM, Fluo-4 AM or Quest Fluo-8™ AM in HHBS at 37°C for 1 hour. The cells were washed twice with 200 µL HHBS, then imaged with a fluorescence microscope (Olympus IX71) using FITC channel.
NOVOStar 96-well data
Fig. 4
Comparisons of Screen Quest™ Fluo-8 NW Calcium Assay Kit, Fluo-4 NW, and Fluo-3 AM in HEK-293 cells were seed overnight in 40,000 cells per 100 µL per well in a 96-well black wall/clear bottom costar plate. The growth medium was removed, and the cells were incubated with 100 µL of Screen Quest™ Fluo-8 NW, Fluo-4 NW, or Flu-3 AM, for 1 hr at 37°C. 30 µM of Carbachol (50 µL/well) was added by NOVOstar (BMG Labtech).
NOVOStar 96-well data
Fig. 5
Carbachol Dose Response in HEK-293. HEK-293 cells were seed overnight in 40,000 per 25 µL per well in growth medium with 1% FBS in a 96-well black wall/clear bottom costar plate. The cells were incubated with 100 µL of the Screen Quest™ Fluo-8 NW Calcium Assay kit, or Fluo-4 NW kit (based on manufacture's protocol) for 1 hour at room temperature. Cabbachol (50 µL/well) was added by NOVOstar (BMG labtech) to achieve the final indicated concentration. The EC50 is about µM which is similar as reported.
FLIPR 384-well data
Fig. 6
ATP Dose Response in HEK-293. HEK-293 cells were seeded overnight in 10,000 cells per 25 µL per well in a 384-well black wall/clear bottom costar plate. The growth medium was removed and the half of the plate was incubated with 25 µL of the Fluo-8 NW Calcium assay kit, and the other half of the plate were incubated with Fluo-4 NW kit (based on manufacture's protocol) for 1 hour at room temperature. ATP (12.5 µL/well) was added by FLIPR 384 to achieve the final indicated concentration. The EC50 is about 4 µM which is similar as reported.
FLIPR 384-well data
Fig. 7
Carbachol Dose Response in HEK-293. HEK -293 cells were seeded overnight in 10,000 cells per 25 µL per wll in a 384-well black wall/clear bottom costar plate. The growth medium was removed and the half of the plate was incubated with 25 µL of the screen Quest™ Fluo-8 NW Calcium Assay kit, and the other half of the plate were incubated with Fluo-4 NW kit for 1 hour at room remperature. Carbachol (12.5 µL/well) was added by FLIPR 384 to achieve the final indicated concentration. The EC50 is about 4 µM which is similar as reported.
NOVOStar 96-well data
Fig. 8
Comparisons of Screen Quest™ Fluo-8 NW Calcium Assay Kit, Fluo-4 NW on Concanavalin A induced Ca entry (capacitative calcium entry (CCE)) in JurKat cells. Jurkat cells were suspended at 4X106 cells per ml in calcium-free HHBS buffer at 2X105 cells/well/100 µL at a 96-well black wall/clear bottom costar plate for 1 hr at37oC, 5% CO2 incubator. At the end of the 10 min incubation, the channel opener Concanavalin A at 1 µg/mL with or without the channel inhibitor SKF96365 at 30 µM were added into the cells. 25 L/well of HBSS with additional 24 mM (5X) Calcium (so final in well concentration of calcium is 5 mM) was added by NOVOstar (BMG Labtech).
Summary
The Screen Quest™ Fluo-8® NW Calcium Assay kit has been optimized for broad range of instruments to give maximum performance with GPCR and calcium channel tagets.
- Brighter: Enable calcium assays with demanding cell lines and receptors;
- 1536 Well-friendly: best Ca2+ indicator for 1536-well Ca2+ assays;
- Less temperature-Sensitive: 37°C & RT loadings generate similar results;
- Larger assay window: Capable of assaying the challenging cell lines and receptors.
Conclusions
The Screen Quest™ Fluo-8® NW Calcium Assay Kit provides an optimized assay method for monitoring G-protein-coupled receptors (GPCRs) and calcium channels. Its ability to generate a very large signal from the weak and robust assay systems enables researchers to have multiple assay chemistries for different receptor and calcium channel targets.
Document: 02.0003.080413r1
Last updated Mon Nov 03 2025
A New Robust No-Wash FLIPR Calcium Assay Kit for Screening GPCR and Calcium Channel Targets